The proposed work will investigate the effects of insulin on the function and structure of the membrane-bound Na-K stimulated, Mg dependent, ouabain sensitive ATP'ase from kidney, brain and adipose tissue. Results from other laboratories and preliminary results from this laboratory indicate that insulin stimulates the ATP'ase under condition of low ATP and Na ion concentration and in the presence of Ca ions. The experimental approach will be to isolate and characterize the enzyme and to study the effects of both Zn containing and Zn-free insulin on the hydrolysis of ATP by both appearance of Pi in the incubation and by the disappearance of ATP. During the characterization of the effects of insulin on the hydrolysis, the partial reactions of the enzyme system will also be studied. The experimental variables will be insulin dose-response, pH dependence, steady state kinetic dependence on (Na ion), (K ion), (Mg), and (ATP), effects on the ouabain sensitive fraction, kinetic parameters in the presence of Ca ions, sugars and amino acids, effects on enzyme preparations of increasing purity, and effects on various pretreatment procedures such as detergents, "chaotropic" ions, organic solvents and enzymatic digestion will be studied.